Small Cationic Antimicrobial Peptides Delocalize Peripheral Membrane Proteins
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Small cationic antimicrobial peptides delocalize PNAS PLUS peripheral membrane proteins Michaela Wenzela, Alina Iulia Chiriacb, Andreas Ottoc, Dagmar Zweytickd, Caroline Maye, Catherine Schumacherf, Ronald Gustg, H. Bauke Albadah, Maya Penkovah, Ute Krämeri, Ralf Erdmannj, Nils Metzler-Nolteh, Suzana K. Strausk, Erhard Bremerl, Dörte Becherc, Heike Brötz-Oesterheltf, Hans-Georg Sahlb, and Julia Elisabeth Bandowa,1 aBiology of Microorganisms, hBioinorganic Chemistry, and iPlant Physiology, eImmune Proteomics, Medical Proteome Center, and jInstitute of Physiological Chemistry, Ruhr University Bochum, 44801 Bochum, Germany; bInstitute for Medical Microbiology, Immunology, and Parasitology, Pharmaceutical Microbiology Section, University of Bonn, 53113 Bonn, Germany; cDepartment of Microbial Physiology and Molecular Biology, Ernst Moritz Arndt University, 17489 Greifswald, Germany; dBiophysics Division, Institute of Molecular Biosciences, University of Graz, 8010 Graz, Austria; fInstitute for Pharmaceutical Biology and Biotechnology, Heinrich Heine University, 40225 Düsseldorf, Germany; gDepartment of Pharmaceutical Chemistry, University of Innsbruck, 6020 Innsbruck, Austria; kDepartment of Chemistry, University of British Columbia, Vancouver, BC, Canada V6T 1Z1; and lDepartment of Biology, University of Marburg, 35037 Marburg, Germany Edited by Michael Zasloff, Georgetown University Medical Center, Washington, DC, and accepted by the Editorial Board February 27, 2014 (received for review October 22, 2013) Short antimicrobial peptides rich in arginine (R) and tryptophan synthetic hexapeptide RWRWRW-NH2 (referred to hereafter as (W) interact with membranes. To learn how this interaction leads “MP196”) (see SI Appendix, Fig. S1 for structure) as a model to bacterial death, we characterized the effects of the minimal representing the minimal pharmacophore of positively charged pharmacophore RWRWRW-NH2. A ruthenium-substituted deriva- and hydrophobic amino acids (16, 17). It is effective against Gram- tive of this peptide localized to the membrane in vivo, and the positive bacteria including methicillin-resistant Staphylococcus peptide also integrated readily into mixed phospholipid bilayers aureus strains, is moderately effective against Gram-negative bac- that resemble Gram-positive membranes. Proteome and Western teria, has low toxicity against human cell lines, and displays low blot analyses showed that integration of the peptide caused de- hemolytic activity (18). MP196 therefore is a promising lead localization of peripheral membrane proteins essential for respi- structure for derivatization and already has yielded peptides with ration and cell-wall biosynthesis, limiting cellular energy and improved activities and altered pharmacological properties (19). MICROBIOLOGY undermining cell-wall integrity. This delocalization phenomenon For example, in a recent systematic L-to-D exchange scan peptides also was observed with the cyclic peptide gramicidin S, indicating with significantly reduced hemolytic activity could be identified (20). the generality of the mechanism. Exogenous glutamate increases Proteomic profiling of the Gram-positive bacterium Bacillus tolerance to the peptide, indicating that osmotic destabilization subtilis exposed to MP196 provided a starting point for mechanistic also contributes to antibacterial efficacy. Bacillus subtilis responds to peptide stress by releasing osmoprotective amino acids, in part Significance via mechanosensitive channels. This response is triggered by mem- brane-targeting bacteriolytic peptides of different structural clas- Multidrug-resistant bacteria present an acute problem to ses as well as by hypoosmotic conditions. medicine, generating interest in novel antimicrobial strategies. Antimicrobial peptides currently are being investigated, both as mechanism of action | respiratory chain | hypoosmotic stress response | antibiotics and as immunomodulatory agents. Many antimicro- metallocenes bial peptides interact with the bacterial membrane, a previously underexplored antibiotic target. We present a system-based study uring the past two decades, bacterial antibiotic resistance has of the mode of action of small cationic peptides and the mecha- Dgrown into a major threat to public health, restoring in- nisms that bacteria use to defend against them. We show that fectious diseases to the list of leading causes of death worldwide peptide integration into the membrane causes delocalization (www.who.int/healthinfo/statistics/bodgbddeathdalyestimates.xls). of essential peripheral membrane proteins. This delocalization As a reaction to this health crisis, several efforts currently are impacts on two cellular processes, namely respiration and cell- underway to discover and develop new natural and synthetic an- wall biosynthesis. We describe a bacterial survival strategy in tibiotic compounds. One promising antibiotic class is antimicrobial which mechanosensitive channels in the bacterial membrane peptides, which occur naturally as part of host defense systems in establish osmoprotection against membrane-targeting bacte- all domains of life (1–4). Antimicrobial peptides range in length riolytic peptides. Understanding the peptides’ mode of action from four to more than 100 amino acids and fall into a number of and bacterial survival strategies opens up new avenues for different structural classes, including α-helical amphiphiles, lipo- devising peptide-based antibacterial strategies. peptides, glycopeptides, lantibiotics, and short cationic peptides. The short cationic peptides offer a potent alternative to longer Author contributions: M.W., H.-G.S., and J.E.B. designed research; M.W., A.I.C., A.O., D.Z., natural antimicrobial peptides. The latter can be difficult to C.M., C.S., and R.G. performed research; H.B.A., M.P., U.K., N.M.-N., S.K.S., E.B., D.B., and H.B.-O. contributed new reagents/analytic tools; M.W., A.I.C., A.O., D.Z., C.M., C.S., isolate and are complicated to synthesize chemically, but short R.G., R.E., H.B.-O., and J.E.B. analyzed data; and M.W., H.B.-O., H.-G.S., and J.E.B. wrote cationic peptides are generated readily by solid-phase peptide the paper. synthesis and are easily accessible for chemical derivatization The authors declare no conflict of interest. (5–8).Theyarecharacterizedbypositivelychargedandby This article is a PNAS Direct Submission. M.Z. is a guest editor invited by the hydrophobic amino acids (9, 10). Previous mechanistic studies Editorial Board. in vitro have examined the interactions of the peptides with Data deposition: The mass spectrometry proteomics data have been deposited to the membranesormembraneextracts(11–15), but their effects on ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository [Vizcaino JA, et al. (2013) The Proteomics Identifications (PRIDE) bacterial physiology have been largely underexplored. database and associated tools: Status in 2013. Nucleic Acids Res 41(D1):D1063–D1069]. A more complete understanding of how these short cationic The dataset ID is PXD000181. antimicrobial peptides bring about bacterial cell death is needed 1To whom correspondence should be addressed. E-mail: [email protected]. to further their optimization and development for practical This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. applications. To achieve this understanding, we studied the 1073/pnas.1319900111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1319900111 PNAS Early Edition | 1of10 Downloaded by guest on September 30, 2021 studies. It identified two major areas for analysis: membrane and S1–S4). Proteins indicative of general cell-envelope stress, mem- cell-wall integrity and energy metabolism. The proteome brane stress, energy limitation, and cell-wall stress were strongly analysis prompted further investigation into the deregulation of up-regulated upon exposure to MP196 (Fig. 1C), indicating that amino acid biosynthesis, which revealed that B. subtilis coun- the cell envelope is the primary target of MP196. From a library teracts the attack on the cell envelope by triggering an osmo- of proteome response profiles that includes the responses to protective release of glutamate. more than 50 antibiotic agents (22–26), several antibiotics were identified that share subsets of these marker proteins with Results MP196, all affecting the bacterial cell envelope (SI Appendix, Inhibition of Macromolecule Biosynthesis Points to the Bacterial Cell Table S5). The strongest overlap in up-regulated proteins was Envelope as a Target of MP196. To explore the inhibition of mac- observed for the membrane-targeting agents gramicidin S and romolecule biosynthesis by MP196 in vivo, we studied incorporation valinomycin. Gramicidin S, a member of the small cationic an- of radioactive precursors. Cells were exposed to MP196 concen- timicrobial peptide class, is structurally distinct from MP196: It trations that reduced growth rates by ∼50% unless noted otherwise is a cyclic amphipathic peptide that does not contain the ar- (SI Appendix,Fig.S2). MP196 moderately inhibited incorporation ginine or tryptophan residues found in MP196. It disrupts of DNA, RNA, protein, and cell-wall precursors (Fig. 1A and membrane function by integrating into the lipid bilayer (27). SI Appendix, Fig. S3). To separate the primary event from sec- Valinomycin is an uncharged dodecadepsipeptide, which acts as ondary effects, the activation of reporter genes was studied in a potassium-carrier ionophore.